White blood cells called T lymphocytes play critical roles in immune defense against viruses, bacteria, fungi, protozoa, and cancer cells. They are also involved in allergies/asthma due to the development of an unwanted or excessive type of immune response to substances (antigens) in our environment and in autoimmune diseases. Because T cells respond to foreign substances (antigens) in the form of peptide-major histocompatibility complex (MHC) molecule pairs on cell surfaces, we wish to know how such complexes interact with specific receptors to evoke the effector activities of mature T cells in the body, as well as regulate their growth, inactivation, differentiation, or death. In particular, we want to understand in molecular detail the protein-protein interactions that turn recognition of antigen by T cells into signals guiding the normal survival and effector functions of these cells, how variations in these recognition and signaling events leads to desirable versus undesirable forms of immunity, and how we can manipulate these events to augment useful immune responses and inhibit damaging ones. Our studies currently focus on biochemical regulatory pathways that help T cells discriminate between self- and foreign peptide:MHC molecule complexes, on the explicit mathematical modeling of these signaling processes in eukaryotic cells, on how the antigen receptors (TCR) of T lymphocytes are selected to provide maximally effective responses to infectious agents, and on how variations in the magnitude and quality of TCR-associated signaling events influence the qualitative nature of adaptive immune responses. We previously reported our analysis of the molecular details of two novel regulatory pathways controlling early signaling by the T cell receptor (SHP-1 phosphatase dependent negative control and MAPK mediated positive control) and a full mathematical computer description of T cell receptor (TCR) signaling that incorporates these novel regulatory pathways. Over the past several years we have focused on using the level of CD5 expression to monitor the affinity of a specific TCR for self-MHC ligands. Using a variety of methods including analysis of partial TCR-associated zeta-chain phosphorylation, we found that we could readily classify T cells according to their self-ligand affinity by this means. Using new methods for assessing the interaction of naive T cells with foreign antigen ligands in the form of oligomers of foreign peptide-MHC class II molecule complexes (pMHC tetramers), we have made the remarkable finding that the affinity of TCRs for self-ligand as determined in the thymus during positive selection directly correlates with the affinity of the T cell for foreign pMHC. In addition, in comparison to other cell surface proteins, CD5 (whose level of expression marks self-pMHC affinity of a T cell) is skewed to the right compared to a log normal distribution. These results indicate that thymic positive selection operates to maximize the affinity of the cells selected into the mature pool, up the to limit set by negative selection, and provide a strong evolutionary rationale for the process of thymic positive selection - maximizing the ability of the mature T cell pool to recognize and respond to foreign antigens. In vivo evidence for the relevance of these findings came from studies with 3 different infectious agents, revealing that CD5hi T cells dominated in the memory pool over CD5 lo cells after resolution of the infectious process, that in older mice, the nave T cell pool is relatively depleted and the memory pool enriched in CD5hi T cells, and that a similar shift between in the fraction of CD5hi T cell can be seen when comparing cord blood and adult samples from humans. These findings are surprising in light of several decades of work on the structural basis of T cell pMHC recognition and the role of peptides vs. MHC molecules in thymic selection and peripheral antigen recognition. Our observations indicate that the field will need to re-visit the interpretations placed on crystal structures of TCR-pMHC interactions to explain the self-foreign antigen affinity connection we have uncovered. These data also have important implications for understanding the nature of T cells responding to self-antigens that promote autoimmune disease and for identifying the best subset of cells to use in adoptive immunotherapy. These findings have been the basis for the development of a new model of repertoire organization in peripheral T cells in which we adopt the M. Cohn concept of a Protection as a repeating unit of specificities that insure an adequate number of responding cells irrespective of body volume; that is, the precursor frequency is similar in mice and humans for a given determinant, but humans have many more cells in absolute number that look at each specificity. This new model also proposes that there is a core or ur repertoire that is based on use of germline gene segments in a TCR. This provides the higher affinity receptors (CD5hi cells) that are best able to recognize foreign antigens, but to prevent pathogen escape from detection, there is a secondary repertoire that diversifies from this germline set using TdT nucleotide additions; these cells on average are less avid for antigen, but provide breadth of coverage. Tests of this model are underway using transcriptome and proteomic analysis of sorted CD5hi and CD5lo T cells as well as using next-gen sequencing of TCR from such sorted T cells.